¹³C metabolic flux analysis identifies an unusual route for pyruvate dissimilation in mycobacteria which requires isocitrate lyase and carbon dioxide fixation.
¹³C metabolic flux analysis identifies an unusual route for pyruvate dissimilation in mycobacteria which requires isocitrate lyase and carbon dioxide fixation.
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DOI：
10.1371/journal.ppat.1002091
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发表时间：
2011-07
期刊：
影响因子：
6.7
通讯作者：
McFadden J
中科院分区：
文献类型：
--
作者：
Beste DJ;Bonde B;Hawkins N;Ward JL;Beale MH;Noack S;Nöh K;Kruger NJ;Ratcliffe RG;McFadden J
结核分枝杆菌需要异柠檬酸裂解酶（ICL）在体内生长和毒力。证明M.结核病也需要ICL在营养物饥饿期间存活，并且在甘油限制的恒化器中的稳态生长期间起作用，这表明该酶的功能超出脂肪代谢。由于异柠檬酸裂解酶是一种潜在的药物靶点，因此阐明该酶的作用非常重要;然而，从未在体内通量水平上研究过异柠檬酸裂解酶的作用。在这里，我们表明，删除两个icl基因之一，损害复制的牛分枝杆菌卡介苗在缓慢的生长速度在碳限制恒化器。为了进一步了解异柠檬酸裂解酶在分枝杆菌中枢代谢中的作用，首次采用13 C-代谢通量分析（MFA）研究了生长速率对分枝杆菌体内代谢通量的影响。用BCG或M.结核病提供13 C标记的甘油或碳酸氢钠。通过测量蛋白质衍生的氨基酸中的13 C同位素异构体标记模式和酶活性测定，我们已经确定了丙酮酸异化的新途径的活性。我们将其命名为GAS途径，因为它利用乙醛酸分流和Anapleurotic反应来氧化丙酮酸，并利用琥珀酰CoA合成酶来产生琥珀酰CoA，并结合通过三羧酸循环的琥珀酸-草酰乙酸片段的非常低的通量。我们确认M.结核病可以将二氧化碳中的碳固定到生物质中。由于人类宿主富含CO2，这一发现需要进一步的体内研究，因为CO2固定可能提供一个可以用新型药物靶向的脆弱点。本研究为进一步研究M.使用13 C-MFA治疗结核病。异柠檬酸裂解酶（ICL）在结核分枝杆菌代谢中的作用已被深入研究，因为证明由该病原体编码的两种异柠檬酸裂解酶基因icl 1和icl 2（尽管一些菌株仅具有icl 1）对于该病原体在宿主中的存活是必需的。这一发现通常被解释为是由于宿主转向消耗脂肪。我们以前证明了当M.结核杆菌（BCG）在碳水化合物上生长缓慢，表明ICL的作用比脂肪代谢更广泛。在这里，我们发现icl 1基因实际上是甘油缓慢生长所必需的。通过喂养BCG和M.结核病细胞同位素标记的碳源，并使用实验和计算分析的组合，我们确定了一种新的代谢途径的碳水化合物代谢，其中ICL是一个关键酶。我们还证明了M.结核病能够从CO2中获得生物质碳。由于CO2在人类宿主中含量丰富，因此可以在开发抗结核病的新型药物治疗中利用CO2固定的抑制。
Mycobacterium tuberculosis requires the enzyme isocitrate lyase (ICL) for growth and virulence in vivo. The demonstration that M. tuberculosis also requires ICL for survival during nutrient starvation and has a role during steady state growth in a glycerol limited chemostat indicates a function for this enzyme which extends beyond fat metabolism. As isocitrate lyase is a potential drug target elucidating the role of this enzyme is of importance; however, the role of isocitrate lyase has never been investigated at the level of in vivo fluxes. Here we show that deletion of one of the two icl genes impairs the replication of Mycobacterium bovis BCG at slow growth rate in a carbon limited chemostat. In order to further understand the role of isocitrate lyase in the central metabolism of mycobacteria the effect of growth rate on the in vivo fluxes was studied for the first time using 13C-metabolic flux analysis (MFA). Tracer experiments were performed with steady state chemostat cultures of BCG or M. tuberculosis supplied with 13C labeled glycerol or sodium bicarbonate. Through measurements of the 13C isotopomer labeling patterns in protein-derived amino acids and enzymatic activity assays we have identified the activity of a novel pathway for pyruvate dissimilation. We named this the GAS pathway because it utilizes the Glyoxylate shunt and Anapleurotic reactions for oxidation of pyruvate, and Succinyl CoA synthetase for the generation of succinyl CoA combined with a very low flux through the succinate – oxaloacetate segment of the tricarboxylic acid cycle. We confirm that M. tuberculosis can fix carbon from CO2 into biomass. As the human host is abundant in CO2 this finding requires further investigation in vivo as CO2 fixation may provide a point of vulnerability that could be targeted with novel drugs. This study also provides a platform for further studies into the metabolism of M. tuberculosis using 13C-MFA. The role of the enzyme isocitrate lyase (ICL) in the metabolism of Mycobacterium tuberculosis has been intensively studied since the demonstration that both of the isocitrate lyase genes encoded by this pathogen, icl1 and icl2 (although some strains only have icl1) are essential for the survival of this pathogen in the host. This finding has been generally interpreted as being due to a shift to the consumption of fats in the host. We previously demonstrated increased ICL activity when the vaccine strain of M. tuberculosis (BCG) was growing slowly on carbohydrates, suggesting a more extended role for ICL than just fat metabolism. Here we show that the gene icl1 is actually essential for slow growth on glycerol. By feeding BCG and M. tuberculosis cells isotopically labeled carbon sources and using a combination of experimental and computational analysis we identified a novel metabolic route for carbohydrate metabolism in which ICL is a key enzyme. We also demonstrated that M. tuberculosis is able to acquire biomass carbon from CO2. As CO2 is abundant in the human host, inhibition of CO2 fixation could be exploited in the development of novel drug treatments against tuberculosis.
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影响因子：
3.2
作者：
Beste, D. J. V.;Laing, E.;McFadden, J. J.
通讯作者：
McFadden, J. J.
影响因子：
2.8
作者：
Gengenbacher, Martin;Rao, Srinivasa P. S.;Dick, Thomas
通讯作者：
Dick, Thomas
影响因子：
4.4
作者：
Gourdon, P;Baucher, MF;Guyonvarch, A
通讯作者：
Guyonvarch, A
影响因子：
5.6
作者：
Goulding, Celia W.;Bowers, Peter M.;Eisenberg, David
通讯作者：
Eisenberg, David
影响因子：
3.2
作者：
FREGNAN, GB;RANDALL, HM;SMITH, DW
通讯作者：
SMITH, DW